PROJECT SUMMARY Autism spectrum disorder (ASD) is a neurodevelopmental condition that affects up to 2% of U.S. children. ASD disabilities have substantial adverse impacts on individuals living with ASD and their families and communities. The estimated annual cost of ASD related disabilities exceeds $250 billion. Recent findings have shown that early detection and intervention can minimize the impact of ASD disabilities, maximize children?s developmental potential, and ultimately improve long-term outcomes. However, no reliable markers currently exist to detect ASD in children under the age of 24 months and the mean age of ASD diagnosis still hovers around age 5. The goal of this study is to address this critical need by evaluating whether early life DNA methylation (DNAm) patterns in blood reflect later ASD diagnosis. Previous studies, by us and others, have found DNAm changes in older children and adults with an ASD diagnosis. However, it isn?t clear whether these differences are present during infancy and may serve as an early life ASD risk biomarker. In addition, despite strong evidence for epigenetic links to ASD most studies have focused on identifying specific loci related to ASD - only one study considered changes in the epigenetic aging pathway and none to our knowledge have examined global DNAm changes related to ASD. Finally, no studies have tested for longitudinal changes in DNAm related to ASD. Here, we overcome previous study limitations by leveraging a unique US population-based study of ASD with infant heal-stick cards (pre- diagnosis) and blood collected at age 5 (post-diagnosis). This enables us to assess prospective and longitudinal DNAm associations with ASD. We use extant (n=968) and newly generated DNAm data on 210 subjects to test our hypotheses that infant blood DNAm patterns are related to later ASD diagnoses and that longitudinal DNAm changes occur concomitant with the emergence of ASD signs. Using samples collected near birth and again at age 5, we will estimate differences: (1A) in heal-stick blood spot DNAm at birth and (1B) in longitudinal DNAm patterns from birth to age 5 between children with and without an ASD diagnosis at age 5. In Aim 2, we will test for epigenetic age acceleration/deceleration: (2A) at birth, and (2B) longitudinally from birth to age 5 between children with and without an ASD diagnosis at age 5. Any significant associations will undergo secondary analyses to test whether the identified DNAm changes are specific to ASD or are also present in SEED children with other developmental disabilities. This R21 mechanism provides the early conceptual support needed to determine whether this area of research warrants further examination in a larger sample, which could include ~4,000 SEED samples. The results of this study may, ultimately, impact child health by providing an early life DNA methylation marker of ASD risk to inform future intervention strategies. Further, the dynamic DNAm changes we identify during early life will likely be important for other childhood health outcomes, more broadly.